Adoptive cell therapy using spherical nucleic acids (snas)

ABSTRACT

The disclosure is related to compositions comprising a cell and a spherical nucleic acid (SNA) comprising a nanoparticle, an oligonucleotide on the surface of the nanoparticle, and an antigen; and to methods for production of such compositions and their applications, including but not limited to adoptive cell therapy.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 62/505,092, filed May 11, 2017, the disclosure of which is incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under U54 CA199091 awarded by the National Institutes of Health. The government has certain rights in the invention.

INCORPORATION BY REFERENCE OF MATERIALS SUBMITTED ELECTRONICALLY

This application contains, as a separate part of the disclosure, a Sequence Listing in computer readable form (Filename: 2017-088_Seqlisting.txt; Size: 460 bytes; Created: May 11, 2018), which is incorporated by reference in its entirety.

BACKGROUND

Spherical nucleic acid (SNA) technology has been demonstrated to be a potent gene regulation and immunostimulatory agent. Current SNA constructs are utilized to deliver therapeutic nucleic acids into the cells. SNAs are a class of nanoconjugates that are overcoming challenges that face current nucleic acid therapies. They provide privileged access at both the cellular and tissue levels. For example, SNAs are actively transported across cell membranes by engaging Class A scavenger receptors [Choi et al., Proc, Natl. Acad. Sci. USA 2013, 110, 7625; Wu et al., J. Am. Chem. Soc. 2014, 136, 7726] while unmodified linear nucleic acids do not enter cells in significant amounts without the use of transfection agents [Luo et al., Nat. Biotechnol. 2000, 18, 33; Opalinska et al., Nat. Rev. Drug Discov. 2002, 1, 503]. In addition, the polyvalent, densely functionalized nucleic acid shell that defines an SNA can act as a high affinity binder for different classes of ligands, including certain receptor proteins [Choi et al., Proc, Natl. Acad. Sci. USA 2013, 110, 7625] and complementary nucleic acid sequences [Lytton-Jean et al., J. Am. Chem. Soc. 2005, 127, 12754]. Consequently, SNAs have emerged as a powerful platform for developing molecular diagnostic probes [Halo et al., Proc. Natl. Acad. Sci. USA 2014, 111, 17104; Prigodich et al., Anal. Chem. 2012, 84, 2062; Zheng et al., Nano Lett. 2009, 9, 3258], and as lead compounds in gene regulation [Jensen et al., Sci. Transl. Med. 2013, 5, 209ra152] and immunomodulation therapies [Radovic-Moreno et al., Proc. Natl. Acad. Sci. USA 2015, 112, 3892; Banga et al., J. Am. Chem. Soc. 2017, 139, 4278]. The three-dimensional architecture of the SNA, rather than the chemical composition of the NP core, is the origin of many of the biochemical properties that make them exceedingly useful in the life sciences and medicine [Choi et al., Proc, Natl. Acad. Sci. USA 2013, 110, 7625; Cutler et al., J. Am. Chem. Soc. 2012, 134, 1376].

Cell-based therapy (e.g., cell-based immunotherapy) is a therapy with curative potential for the treatment of cancer. T cells and other immune cells may be modified to target tumor antigens through the introduction of an antigen that is specific to a tumor antigen.

SUMMARY

Disclosed herein are spherical nucleic acid (SNA) materials (i.e., structures consisting of a nanoparticle core, oligonucleotides, and other possible classes of molecule—including peptide or protein antigens) in a cell-based therapy. In some embodiments of the present disclosure, the SNAs are first used to treat a subset of cells ex vivo before reinfusion into the animal or patient; this key step allows for a controlled environment for the SNAs to interact with cells, and is advantageous compared with a system in which distribution of SNAs throughout the body occurs following injection of SNAs directly into a patient subcutaneously or intravenously via the blood stream. The types of cells contemplated by the disclosure include, but are not limited to T-cells (e.g., CD4, CD8, and gamma delta), natural killer (NK) cells, B-cells, macrophages, and dendritic cells.

In some embodiments, after the SNAs are allowed to enter and activate the cells, the SNA-loaded cells are then re-infused into animals, at which time the active agent becomes the cells themselves. These SNA-loaded cells perform multiple functions in an immunotherapeutic mode of action. Among the demonstrated activities of these cells, once re-introduced to animal models, are 1) the transfer of SNA and SNA components (adjuvant polynucleotides (e.g., a CpG oligonucleotide), antigens) to other cells (such as an antigen presenting cell (APC)); and 2) direct attack of target cells.

The method of using SNAs to enter and activate cells outside the body, as disclosed herein, enables superior activation of these cells and targeting to the tumor site and lymph system only. This avoids the potential problems of SNA distribution to non-target organs.

In various embodiments, the methods of the disclosure are used to treat cancer. In related embodiments, the cancer is a hematological tumor or a solid tumor. In still further embodiments, the cancer is bladder cancer, brain cancer, cervical cancer, colon/rectal cancer, leukemia, lymphoma, liver cancer, ovarian cancer, pancreatic cancer, sarcoma, prostate cancer, or breast cancer.

In some aspects, the disclosure provides a composition comprising a pharmaceutically acceptable carrier and a cell having a spherical nucleic acid (SNA) contained therein, wherein the cell is obtained from an individual and the SNA comprises a nanoparticle, an oligonucleotide on the surface of the nanoparticle, and an antigen. In some embodiments, the antigen is a prostate-specific antigen (PSA) peptide, mesothelin, glycoprotein 100 (gp100), prostate specific membrane antigen (PSMA), or prostatic acid phosphatase (PAP). In further embodiments, the nanoparticle is a liposome. In some embodiments, the liposome comprises a lipid selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC), 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DSPG), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE), cardiolipin, and lipid A.

In further embodiments, the oligonucleotide comprises a tocopherol, a cholesterol moiety, DOPE-butamide-phenylmaleimido, or lyso-phosphoethanolamine-butamide-pneylmaleimido. In some embodiments, the oligonucleotide comprises RNA or DNA. In still further embodiments, the oligonucleotide comprises a sequence that is a toll-like receptor (TLR) agonist. In some embodiments, the TLR is chosen from the group consisting of toll-like receptor 1 (TLR1), toll-like receptor 2 (TLR2), toll-like receptor 3 (TLR3), toll-like receptor 4 (TLR4), toll-like receptor 5 (TLR5), toll-like receptor 6 (TLR6), toll-like receptor 7 (TLR7), toll-like receptor 8 (TLR8), toll-like receptor 9 (TLR9), toll-like receptor 10 (TLR10), toll-like receptor 11 (TLR11), toll-like receptor 12 (TLR12), and toll-like receptor 13 (TLR13). In some embodiments, the oligonucleotide comprises a CpG nucleotide sequence.

In some embodiments, the composition further comprises an additional oligonucleotide. In further embodiments, the additional oligonucleotide comprises RNA or DNA.

In still further embodiments, the RNA is a non-coding RNA. In some embodiments, the non-coding RNA is an inhibitory RNA (RNAi). In further embodiments, the RNAi is selected from the group consisting of a small inhibitory RNA (siRNA), a single-stranded RNA (ssRNA) that forms a triplex with double stranded DNA, and a ribozyme. In some embodiments, the RNA is a microRNA. In further embodiments, the DNA is antisense-DNA.

In some embodiments, the nanoparticle has a diameter of 50 nanometers or less. In further embodiments, the SNA comprises about 10 to about 80 double stranded oligonucleotides. In some embodiments, the SNA comprises 70 double stranded oligonucleotides.

In some embodiments, the cell is a T-cell, a natural killer (NK) cell, a B-cell, a macrophage, a dendritic cell, or a combination thereof. In further embodiments, the antigen is encapsulated in the nanoparticle. In some embodiments, the antigen is on the surface of the nanoparticle.

In some aspects, the disclosure provides a method of making a composition of the disclosure comprising contacting the cell with the SNA to form the composition. In some embodiments, the cell is obtained from an individual in need of adoptive cell therapy.

In some aspects, the disclosure provides a vaccine comprising a composition of the disclosure, and an adjuvant.

In some aspects, the disclosure provides a method of treating an individual in need of adoptive cell therapy comprising administering a composition of the disclosure to the individual.

In some aspects, a method of producing an immune response to cancer in an individual is provided, comprising administering to the individual an effective amount of a composition of the disclosure, or a vaccine of the disclosure, thereby producing an immune response to cancer in the individual. In some embodiments, the immune response is a neutralizing antibody response or a protective antibody response.

In some aspects, the disclosure provides a method of immunizing an individual against cancer comprising administering to the individual an effective amount of a composition or a vaccine of the disclosure, thereby immunizing the individual against cancer. In some embodiments, the cancer is selected from the group consisting of prostate, breast, melanoma, and lung cancer.

In some aspects, a method of inhibiting expression of a gene is provided comprising hybridizing a polynucleotide encoding the gene with one or more oligonucleotides complementary to all or a portion of the polynucleotide, the oligonucleotide being the additional oligonucleotide of a composition of the disclosure, wherein hybridizing between the polynucleotide and the oligonucleotide occurs over a length of the polynucleotide with a degree of complementarity sufficient to inhibit expression of the gene product. In some embodiments, expression of the gene product is inhibited in vivo. In further embodiments, expression of the gene product is inhibited in vitro. In still further embodiments, the gene is PD-1 or PD-1.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts various SNAs, showing that the SNAs can vary both in composition and in structure.

FIG. 2 is a schematic depicting the immunostimulatory effects an SNA can have following endocytosis.

FIG. 3 is a schematic illustrating the use of SNAs as a vaccine for cancer.

FIG. 4 is a schematic depicting the use of SNAs in T-cell chaperone cellular therapy.

FIG. 5 shows that T chaperones are capable of priming antigen specific CD8+ T cells in vivo. A) Spleen B) Lymph Node. On day zero 3×10⁶ naïve CD45.1⁺PMEL T cells were stained with e450 cell proliferation dye and then transferred i.v. into WT C57BL/6 mice. The next day 5×10⁶ T chaperones were transferred i.v. after incubation with either admix CpG/gp100 controls or SNAs overnight at 4 μg/mL of gp100 and 1 μM CpG. On day 6 CD45.1⁺ T cells were isolated from spleen and inguinal lymph nodes and analyzed for proliferation and IFN-γ production by flow cytometry.

FIG. 6 demonstrates that SNA derived T chaperones promote durable killing function and tumor protection. A) In vitro killing by T chap_(ISNA). Purified GD8⁺PMEL T cells were incubated with the labeled conditions overnight, washed and allowed to rest for four days. Overnight controls were incubated one day prior to the assay being performed and plated with B16 cells on the same day as the four day rest group. Before incubation, tumor cells were labeled with e450 dye and gated as CD45-e450⁺ for analysis of apoptotic markers by flow cytometry. For activation of T cells prior to killing assay αCD3/αCD28 was given at 1 μg/mL, gp100 was given at 4 μg/ml, CpG was at 1 μM. Experiment was plated at a 50:1 ratio of T cell: tumor cell for 12 hours. B) T chap_(ISNA) control B16 tumor growth. C57BL/6 were challenged subcutaneously with 1×10⁶ B16-F10 tumor cells on the hind flank. On day 8, when tumors were approximately 150 mm³, mice were intravenously administered 200 μL of PBS or 10⁶ T chaps in 200 μL of PBS.

FIG. 7 shows that SNA T chaperones control LLC1-OVA tumors and increase polyfunctional CD8+ T cell tumor infiltrates. A) C57BL/6 were challenged subcutaneously with 1×10⁶ LLC1-OVA tumor cells on the hind flank. On day 8, when tumors were approximately 150 mm³, mice were intravenously administered 200 μL of PBS or 1×10⁶ T chaperones in 200 μL of PBS. T chaperones were prepared by incubating 0.5×10⁶ OT-1 CD8⁺ T cells in 200 μL volume overnight with 0.06 μg/mL of OVA-1 peptide and 1 μM of CpG. Cells were washed three times before transfer B) T chaperones promote tumor infiltration of polyfunctional T cells. When control tumors reached growth limit, on day 23, tumor tissue was analyzed for CD8⁺ T cell infiltrates by flow cytometry for cytokine production.

FIG. 8 shows that SNAs induce exosomal antigen transfer for bystander priming function. A) ISNA T chap exosomes transfer antigen to T cells and DCs in vitro. PMEL CD8⁺ T cells were incubated with labeled conditions overnight. The next day they were washed and seeded in the top portion of a transwell system with 0.4 μm pores. The bottom wells contained naïve CD8⁺ PMEL T cells or naïve DCs. After 24 hours the cells from the bottom wells were isolated and used in priming assays with naïve e450 stained CD8⁺ CD45.1⁺ PMEL T cells. T-APCs and DCs were plated at a 1:1 ratio with naïve responder cells. After four days, CD45.1⁺ cells were analyzed for activation markers using flow cytometry. B) ISNA T chap exosomes directly activate PMEL CD8⁺ T cells. Isolated T cells were incubated with the indicated conditions overnight. The next day they were washed and re-plated with exosome free medium. 10 μg of exosomes were then added directly to naïve e450 stained CD8⁺ for four days. Cells were then analyzed by flow cytometry. C) ISNA T chap exosomes induce T cell priming in vivo. On day 0 naïve e450 labeled CD8⁺ T cells were intravenously transferred into WT mice. On day 1, 30 μg of T cell derived exosomes were transferred intravenously. On day 6 Vβ13⁺ e450⁺ CD8⁺ T cells were examined for proliferation by flow cytometry.

DETAILED DESCRIPTION

The present disclosure is directed to compositions comprising SNAs and their use in cell-based therapies. There are several advantages to using SNAs in cell-based therapies. For example, the 3-D structure of the SNA creates a platform for vast combinations of oligonucleotide shell decoration and protein (e.g., antibodies, peptides, cytokines) encapsulation within the core. SNAs of the disclosure load material into T-cells ex vivo to create T-cell chaperones. This flexibility allows for the creation of personalized T-cell therapy via SNA design. In some embodiments, the antigen core of the SNA guides the attack against different types of cancer based on the expression of tumor associated antigens. The endocytosis of SNAs allows for non-viral modification of T-cells to create multifunctional T-cell chaperones. T-cell chaperones transfer SNA derived adjuvant and antigen cargo to APCs in vivo to boost priming function and directly kill tumor cells. T-cell chaperones efficiently home to immune priming sites, including draining lymph nodes and tumor tissue. T-chaperone bystander transfer eliminates the need for systemic administration regimens, decreases the amount of SNA material needed, and prevents off-target side effects associated with systemic drugs.

The terms “polynucleotide” and “oligonucleotide” are interchangeable as used herein.

The term “T cell chaperone” or “T chap” refers to a cell that has been contacted with a SNA of the disclosure, in order to load the cell with an immunostimulatory oligonucleotide and a tumor-associated antigen.

An “immune response” is a response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus, such as a pathogen or antigen (e.g., formulated as an antigenic composition or a vaccine). An immune response can be a B cell response, which results in the production of specific antibodies, such as antigen specific neutralizing antibodies. An immune response can also be a T cell response, such as a CD4⁺ response or a CD8⁺ response. B cell and T cell responses are aspects of a “cellular” immune response. An immune response can also be a “humoral” immune response, which is mediated by antibodies. In some cases, the response is specific for a particular antigen (that is, an “antigen-specific response”). An immune response can be measured, for example, by ELISA-neutralization assay. Exposure of a subject to an immunogenic stimulus, such as an antigen (e.g., formulated as an antigenic composition or vaccine), elicits a primary immune response specific for the stimulus, that is, the exposure “primes” the immune response.

As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

Spherical Nucleic Acids. Spherical nucleic acids (SNAs) comprise densely functionalized and highly oriented polynucleotides on the surface of a nanoparticle which can either be organic (e.g., a liposome) inorganic (such as gold, silver, or platinum) or hollow (e.g., silica-based). The spherical architecture of the polynucleotide shell confers unique advantages over traditional nucleic acid delivery methods, including entry into nearly all cells independent of transfection agents and resistance to nuclease degradation. Furthermore, SNAs can penetrate biological barriers, including the blood-brain and blood-tumor barriers as well as the epidermis.

See FIG. 1 . SNAs are useful in a myriad of biomedical applications, including as gene regulatory therapeutics (where SNAs are formulated with antisense and/or siRNA oligonucleotides) and as immunostimulatory (IS) therapeutics (where SNAs are formulated with immunostimulatory oligonucleotides and can induce an immunotherapeutic response—see FIGS. 2-4 ). As a structural class, immunostimulatory SNAs (IS-SNAs) efficiently enter endosomes and stimulate immune system signaling via, e.g., toll-like receptor 9 (TLR9), TLR3, and/or TLR7/8). FIG. 3 depicts the use of SNAs as a vaccine for cancer, while FIG. 4 illustrates the use of SNAs for T-cell-based therapy. When used as an enabler for T-cell-based therapy, the initial interaction of the SNAs is with T-cells rather than antigen-presenting cells (APCs). Also, the interaction between SNAs and T-cells takes place ex vivo, and T-cells loaded with SNAs are then re-administered to the subject. The immune response is then generated by the T-cells containing SNAs.

Nanoparticles are therefore provided which are functionalized to have a polynucleotide attached thereto. In general, nanoparticles contemplated include any compound or substance with a high loading capacity for a polynucleotide as described herein, including for example and without limitation, a metal, a semiconductor, a liposomal particle, insulator particle compositions, and a dendrimer (organic versus inorganic).

Thus, nanoparticles are contemplated which comprise a variety of inorganic materials including, but not limited to, metals, semi-conductor materials or ceramics as described in U.S. Patent Publication No 20030147966. For example, metal-based nanoparticles include those described herein. Ceramic nanoparticle materials include, but are not limited to, brushite, tricalcium phosphate, alumina, silica, and zirconia. Organic materials from which nanoparticles are produced include carbon. Nanoparticle polymers include polystyrene, silicone rubber, polycarbonate, polyurethanes, polypropylenes, polymethylmethacrylate, polyvinyl chloride, polyesters, polyethers, and polyethylene. Biodegradable, biopolymer (e.g., polypeptides such as BSA, polysaccharides, etc.), other biological materials (e.g., carbohydrates), and/or polymeric compounds are also contemplated for use in producing nanoparticles.

Liposomal particles, for example as disclosed in International Patent Application No. PCT/US2014/068429 (incorporated by reference herein in its entirety, particularly with respect to the discussion of liposomal particles) are also contemplated by the disclosure. Hollow particles, for example as described in U.S. Patent Publication Number 2012/0282186 (incorporated by reference herein in its entirety) are also contemplated herein. Liposomal particles of the disclosure have at least a substantially spherical geometry, an internal side and an external side, and comprise a lipid bilayer. The lipid bilayer comprises, in various embodiments, a lipid from the phosphocholine family of lipids or the phosphoethanolamine family of lipids. While not meant to be limiting, the first-lipid is chosen from group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC), 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DSPG), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE), cardiolipin, lipid A, and a combination thereof.

In one embodiment, the nanoparticle is metallic, and in various aspects, the nanoparticle is a colloidal metal. Thus, in various embodiments, nanoparticles useful in the practice of the methods include metal (including for example and without limitation, gold, silver, platinum, aluminum, palladium, copper, cobalt, indium, nickel, or any other metal amenable to nanoparticle formation), semiconductor (including for example and without limitation, CdSe, CdS, and CdS or CdSe coated with ZnS) and magnetic (for example, ferromagnetite) colloidal materials. Other nanoparticles useful in the practice of the invention include, also without limitation, ZnS, ZnO, Ti, TiO2, Sn, SnO2, Si, SiO₂, Fe, Fe+4, Ag, Cu, Ni, Al, steel, cobalt-chrome alloys, Cd, titanium alloys, AgI, AgBr, HgI2, PbS, PbSe, ZnTe, CdTe, In2S3, In2Se3, Cd3P2, Cd3As2, InAs, and GaAs. Methods of making ZnS, ZnO, TiO2, AgI, AgBr, HgI2, PbS, PbSe, ZnTe, CdTe, In2S3, In2Se3, Cd3P2, Cd3As2, InAs, and GaAs nanoparticles are also known in the art. See, e.g., Weller, Angew. Chem. Int. Ed. Engl., 32, 41 (1993); Henglein, Top. Curr. Chem., 143, 113 (1988); Henglein, Chem. Rev., 89, 1861 (1989); Brus, Appl. Phys. A., 53, 465 (1991); Bahncmann, in Photochemical Conversion and Storage of Solar Energy (eds. Pelizetti and Schiavello 1991), page 251; Wang and Herron, J. Phys. Chem., 95, 525 (1991); Olshavsky, et al., J. Am. Chem. Soc., 112, 9438 (1990); Ushida et al., J. Phys. Chem., 95, 5382 (1992).

In practice, methods of increasing cellular uptake and inhibiting gene expression are provided using any suitable particle having oligonucleotides attached thereto that do not interfere with complex formation, i.e., hybridization to a target polynucleotide. The size, shape and chemical composition of the particles contribute to the properties of the resulting oligonucleotide-functionalized nanoparticle. These properties include for example, optical properties, optoelectronic properties, electrochemical properties, electronic properties, stability in various solutions, magnetic properties, and pore and channel size variation. The use of mixtures of particles having different sizes, shapes and/or chemical compositions, as well as the use of nanoparticles having uniform sizes, shapes and chemical composition, is contemplated. Examples of suitable particles include, without limitation, nanoparticles particles, aggregate particles, isotropic (such as spherical particles) and anisotropic particles (such as non-spherical rods, tetrahedral, prisms) and core-shell particles such as the ones described in U.S. patent application Ser. No. 10/034,451, filed Dec. 28, 2002, and International Application No. PCT/US01/50825, filed Dec. 28, 2002, the disclosures of which are incorporated by reference in their entirety.

Methods of making metal, semiconductor and magnetic nanoparticles are well-known in the art. See, for example, Schmid, G. (ed.) Clusters and Colloids (VCH, Weinheim, 1994); Hayat, M. A. (ed.) Colloidal Gold: Principles, Methods, and Applications (Academic Press, San Diego, 1991); Massart, R., IEEE Transactions On Magnetics, 17, 1247 (1981); Ahmadi, T. S. et al., Science, 272, 1924 (1996); Henglein, A. et al., J. Phys. Chem., 99, 14129 (1995); Curtis, A. C., et al., Angew. Chem. Int. Ed. Engl., 27, 1530 (1988). Preparation of polyalkylcyanoacrylate nanoparticles prepared is described in Fattal, et al., J. Controlled Release (1998) 53: 137-143 and U.S. Pat. No. 4,489,055. Methods for making nanoparticles comprising poly(D-glucaramidoamine)s are described in Liu, et al., J. Am. Chem. Soc. (2004) 126:7422-7423. Preparation of nanoparticles comprising polymerized methylmethacrylate (MMA) is described in Tondelli, et al., Nucl. Acids Res. (1998) 26:5425-5431, and preparation of dendrimer nanoparticles is described in, for example Kukowska-Latallo, et al., Proc. Natl. Acad. Sci. USA (1996) 93:4897-4902 (Starburst polyamidoamine dendrimers)

Suitable nanoparticles are also commercially available from, for example, Ted Pella, Inc. (gold), Amersham Corporation (gold) and Nanoprobes, Inc. (gold).

Also as described in US Patent Publication No. 20030147966, nanoparticles comprising materials described herein are available commercially or they can be produced from progressive nucleation in solution (e.g., by colloid reaction), or by various physical and chemical vapor deposition processes, such as sputter deposition. See, e.g., HaVashi, (1987) Vac. Sci. Technol. July/August 1987, A5(4):1375-84; Hayashi, (1987) Physics Today, December 1987, pp. 44-60; MRS Bulletin, January 1990, pgs. 16-47.

As further described in U.S. Patent Publication No. 20030147966, nanoparticles contemplated are produced using HAuCl₄ and a citrate-reducing agent, using methods known in the art. See, e.g., Marinakos et al., (1999) Adv. Mater. 11: 34-37; Marinakos et al., (1998) Chem. Mater. 10: 1214-19; Enustun & Turkevich, (1963) J. Am. Chem. Soc. 85: 3317. Tin oxide nanoparticles having a dispersed aggregate particle size of about 140 nm are available commercially from Vacuum Metallurgical Co., Ltd. of Chiba, Japan. Other commercially available nanoparticles of various compositions and size ranges are available, for example, from Vector Laboratories, Inc. of Burlingame, Calif.

Nanoparticles can range in size from about 1 nm to about 250 nm in mean diameter, about 1 nm to about 240 nm in mean diameter, about 1 nm to about 230 nm in mean diameter, about 1 nm to about 220 nm in mean diameter, about 1 nm to about 210 nm in mean diameter, about 1 nm to about 200 nm in mean diameter, about 1 nm to about 190 nm in mean diameter, about 1 nm to about 180 nm in mean diameter, about 1 nm to about 170 nm in mean diameter, about 1 nm to about 160 nm in mean diameter, about 1 nm to about 150 nm in mean diameter, about 1 nm to about 140 nm in mean diameter, about 1 nm to about 130 nm in mean diameter, about 1 nm to about 120 nm in mean diameter, about 1 nm to about 110 nm in mean diameter, about 1 nm to about 100 nm in mean diameter, about 1 nm to about 90 nm in mean diameter, about 1 nm to about 80 nm in mean diameter, about 1 nm to about 70 nm in mean diameter, about 1 nm to about 60 nm in mean diameter, about 1 nm to about 50 nm in mean diameter, about 1 nm to about 40 nm in mean diameter, about 1 nm to about 30 nm in mean diameter, or about 1 nm to about 20 nm in mean diameter, about 1 nm to about 10 nm in mean diameter. In other aspects, the size of the nanoparticles is from about 5 nm to about 150 nm (mean diameter), from about 5 to about 50 nm, from about 10 to about 30 nm, from about 10 to 150 nm, from about 10 to about 100 nm, or about 10 to about 50 nm. The size of the nanoparticles is from about 5 nm to about 150 nm (mean diameter), from about 30 to about 100 nm, from about 40 to about 80 nm. The size of the nanoparticles used in a method varies as required by their particular use or application. The variation of size is advantageously used to optimize certain physical characteristics of the nanoparticles, for example, optical properties or the amount of surface area that can be functionalized as described herein. In further embodiments, a plurality of SNAs (e.g., liposomal particles) is produced and the SNAs in the plurality have a mean diameter of less than or equal to about 50 nanometers (e.g., about 5 nanometers to about 50 nanometers, or about 5 nanometers to about 40 nanometers, or about 5 nanometers to about 30 nanometers, or about 5 nanometers to about 20 nanometers, or about 10 nanometers to about 50 nanometers, or about 10 nanometers to about 40 nanometers, or about 10 nanometers to about 30 nanometers, or about 10 nanometers to about 20 nanometers). In further embodiments, the SNAs in the plurality created by a method of the disclosure have a mean diameter of less than or equal to about 20 nanometers, or less than or equal to about 25 nanometers, or less than or equal to about 30 nanometers, or less than or equal to about 35 nanometers, or less than or equal to about 40 nanometers, or less than or equal to about 45 nanometers.

Antigen. The present disclosure provides SNAs comprising an antigen. In various embodiments, the antigen is a tumor-associated antigen. In some embodiments, the antigen is a prostate-specific antigen (PSA) peptide, mesothelin, glycoprotein 100 (gp100), prostate specific membrane antigen (PSMA), or prostatic acid phosphatase (PAP). Other antigens are contemplated for use according to the compositions and methods of the disclosure; any antigen for which an immune response is desired is contemplated herein.

It is contemplated herein that an antigen for use in the compositions and methods of the disclosure are encapsulated with a SNA, or an antigen is on the surface of the SNA, or both.

Cells. The disclosure contemplates contacting a SNA as described herein with a cell in order to load the cell with an immunestimulatory oligonucleotide and a tumor-associated antigen. Cells contemplated for use in the compositions and methods of the disclosure include, but are not limited to, a T-cell, a natural killer (NK) cell, a B-cell, a macrophage, a dendritic cell, or a combination thereof.

Polynucleotides. The term “nucleotide” or its plural as used herein is interchangeable with modified forms as discussed herein and otherwise known in the art. In certain instances, the art uses the term “nucleobase” which embraces naturally-occurring nucleotide, and non-naturally-occurring nucleotides which include modified nucleotides. Thus, nucleotide or nucleobase means the naturally occurring nucleobases A, G, C, T, and U. Non-naturally occurring nucleobases include, for example and without limitations, xanthine, diaminopurine, 8-oxo-N6-methyladenine, 7-deazaxanthine, 7-deazaguanine, N4,N4-ethanocytosin, N′,N′-ethano-2,6-diaminopurine, 5-methylcytosine (mC), 5-(C3-C6)-alkynyl-cytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-tr-iazolopyridin, isocytosine, isoguanine, inosine and the “non-naturally occurring” nucleobases described in Benner et al., U.S. Pat. No. 5,432,272 and Susan M. Freier and Karl-Heinz Altmann, 1997, Nucleic Acids Research, vol. 25: pp 4429-4443. The term “nucleobase” also includes not only the known purine and pyrimidine heterocycles, but also heterocyclic analogues and tautomers thereof. Further naturally and non-naturally occurring nucleobases include those disclosed in U.S. Pat. No. 3,687,808 (Merigan, et al.), in Chapter 15 by Sanghvi, in Antisense Research and Application, Ed. S. T. Crooke and B. Lebleu, CRC Press, 1993, in Englisch et al., 1991, Angewandte Chemie, International Edition, 30: 613-722 (see especially pages 622 and 623, and in the Concise Encyclopedia of Polymer Science and Engineering, J. I. Kroschwitz Ed., John Wiley & Sons, 1990, pages 858-859, Cook, Anti-Cancer Drug Design 1991, 6, 585-607, each of which are hereby incorporated by reference in their entirety). In various aspects, polynucleotides also include one or more “nucleosidic bases” or “base units” which are a category of non-naturally-occurring nucleotides that include compounds such as heterocyclic compounds that can serve like nucleobases, including certain “universal bases” that are not nucleosidic bases in the most classical sense but serve as nucleosidic bases. Universal bases include 3-nitropyrrole, optionally substituted indoles (e.g., 5-nitroindole), and optionally substituted hypoxanthine. Other desirable universal bases include, pyrrole, diazole or triazole derivatives, including those universal bases known in the art.

Modified nucleotides are described in EP 1 072 679 and WO 97/12896, the disclosures of which are incorporated herein by reference. Modified nucleobases include without limitation, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified bases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzox-azin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified bases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Additional nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., 1991, Angewandte Chemie, International Edition, 30: 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of these bases are useful for increasing the binding affinity and include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. and are, in certain aspects combined with 2′-O-methoxyethyl sugar modifications. See, U.S. Pat. Nos. 3,687,808, 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; 5,750,692 and 5,681,941, the disclosures of which are incorporated herein by reference.

Methods of making polynucleotides of a predetermined sequence are well-known. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed. 1989) and F. Eckstein (ed.) Oligonucleotides and Analogues, 1st Ed. (Oxford University Press, New York, 1991). Solid-phase synthesis methods are preferred for both polyribonucleotides and polydeoxyribonucleotides (the well-known methods of synthesizing DNA are also useful for synthesizing RNA). Polyribonucleotides can also be prepared enzymatically. Non-naturally occurring nucleobases can be incorporated into the polynucleotide, as well. See, e.g., U.S. Pat. No. 7,223,833; Katz, J. Am. Chem. Soc., 74:2238 (1951); Yamane, et al., J. Am. Chem. Soc., 83:2599 (1961); Kosturko, et al., Biochemistry, 13:3949 (1974); Thomas, J. Am. Chem. Soc., 76:6032 (1954); Zhang, et al., J. Am. Chem. Soc., 127:74-75 (2005); and Zimmermann, et al., J. Am. Chem. Soc., 124:13684-13685 (2002).

Nanoparticles provided that are functionalized with a polynucleotide, or a modified form thereof generally comprise a polynucleotide from about 5 nucleotides to about 100 nucleotides in length. More specifically, nanoparticles are functionalized with a polynucleotide that is about 5 to about 90 nucleotides in length, about 5 to about 80 nucleotides in length, about 5 to about 70 nucleotides in length, about 5 to about 60 nucleotides in length, about 5 to about 50 nucleotides in length about 5 to about 45 nucleotides in length, about 5 to about 40 nucleotides in length, about 5 to about 35 nucleotides in length, about 5 to about 30 nucleotides in length, about 5 to about 25 nucleotides in length, about 5 to about 20 nucleotides in length, about 5 to about 15 nucleotides in length, about 5 to about 10 nucleotides in length, and all polynucleotides intermediate in length of the sizes specifically disclosed to the extent that the polynucleotide is able to achieve the desired result. Accordingly, polynucleotides of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, about 125, about 150, about 175, about 200, about 250, about 300, about 350, about 400, about 450, about 500 or more nucleotides in length are contemplated.

In some embodiments, the polynucleotide attached to a nanoparticle is DNA. When DNA is attached to the nanoparticle, the DNA is in some embodiments comprised of a sequence that is sufficiently complementary to a target region of a polynucleotide such that hybridization of the DNA polynucleotide attached to a nanoparticle and the target polynucleotide takes place, thereby associating the target polynucleotide to the nanoparticle. The DNA in various aspects is single stranded or double-stranded, as long as the double-stranded molecule also includes a single strand region that hybridizes to a single strand region of the target polynucleotide. In some aspects, hybridization of the polynucleotide functionalized on the nanoparticle can form a triplex structure with a double-stranded target polynucleotide. In another aspect, a triplex structure can be formed by hybridization of a double-stranded oligonucleotide functionalized on a nanoparticle to a single-stranded target polynucleotide.

In some embodiments, the disclosure contemplates that a polynucleotide attached to a nanoparticle is RNA. The RNA can be either single-stranded or double-stranded, so long as it is able to hybridize to a target polynucleotide.

In some aspects, multiple polynucleotides are functionalized to a nanoparticle. In various aspects, the multiple polynucleotides each have the same sequence, while in other aspects one or more polynucleotides have a different sequence. In further aspects, multiple polynucleotides are arranged in tandem and are separated by a spacer. Spacers are described in more detail herein below.

Polynucleotide attachment to a nanoparticle. Polynucleotides contemplated for use in the methods include those bound to the nanoparticle through any means (e.g., covalent or non-covalent attachment). Regardless of the means by which the polynucleotide is attached to the nanoparticle, attachment in various aspects is effected through a 5′ linkage, a 3′ linkage, some type of internal linkage, or any combination of these attachments. In some embodiments, the polynucleotide is covalently attached to a nanoparticle. In further embodiments, the polynucleotide is non-covalently attached to a nanoparticle. An oligonucleotide of the disclosure comprises, in various embodiments, a tocopherol, a cholesterol moiety, DOPE-butamide-phenylmaleimido, or lyso-phosphoethanolamine-butamide-pneylmaleimido. See also U.S. Patent Application Publication No. 2016/0310425, incorporated by reference herein in its entirety.

Methods of attachment are known to those of ordinary skill in the art and are described in US Publication No. 2009/0209629, which is incorporated by reference herein in its entirety. Methods of attaching RNA to a nanoparticle are generally described in PCT/US2009/65822, which is incorporated by reference herein in its entirety. Methods of associating polynucleotides with a liposomal particle are described in PCT/US2014/068429, which is incorporated by reference herein in its entirety.

Spacers. In certain aspects, functionalized nanoparticles are contemplated which include those wherein an oligonucleotide is attached to the nanoparticle through a spacer. “Spacer” as used herein means a moiety that does not participate in modulating gene expression per se but which serves to increase distance between the nanoparticle and the functional oligonucleotide, or to increase distance between individual oligonucleotides when attached to the nanoparticle in multiple copies. Thus, spacers are contemplated being located between individual oligonucleotides in tandem, whether the oligonucleotides have the same sequence or have different sequences. In some aspects, the spacer when present is an organic moiety. In another aspect, the spacer is a polymer, including but not limited to a water-soluble polymer, a polynucleotide, a polypeptide, an oligosaccharide, a carbohydrate, a lipid, an ethylglycol, or combinations thereof.

In certain aspects, the polynucleotide has a spacer through which it is covalently bound to the nanoparticles. As a result of the binding of the spacer to the nanoparticles, the polynucleotide is spaced away from the surface of the nanoparticles and is more accessible for hybridization with its target. In various embodiments, the length of the spacer is or is equivalent to at least about 5 nucleotides, 5-10 nucleotides, 10 nucleotides, 10-30 nucleotides, or even greater than 30 nucleotides. The spacer may have any sequence which does not interfere with the ability of the polynucleotides to become bound to the nanoparticles or to the target polynucleotide. In certain aspects, the bases of a polynucleotide spacer are all adenylic acids, all thymidylic acids, all cytidylic acids, all guanylic acids, all uridylic acids, or all some other modified base.

Nanoparticle surface density. A surface density adequate to make the nanoparticles stable and the conditions necessary to obtain it for a desired combination of nanoparticles and polynucleotides can be determined empirically. Generally, a surface density of at least about 2 pmoles/cm² will be adequate to provide stable nanoparticle-oligonucleotide compositions. In some aspects, the surface density is at least 15 pmoles/cm². Methods are also provided wherein the polynucleotide is bound to the nanoparticle at a surface density of at least 2 pmol/cm², at least 3 pmol/cm², at least 4 pmol/cm², at least 5 pmol/cm², at least 6 pmol/cm², at least 7 pmol/cm², at least 8 pmol/cm², at least 9 pmol/cm², at least 10 pmol/cm², at least about 15 pmol/cm2, at least about 19 pmol/cm², at least about 20 pmol/cm², at least about 25 pmol/cm², at least about 30 pmol/cm², at least about 35 pmol/cm², at least about 40 pmol/cm², at least about 45 pmol/cm², at least about 50 pmol/cm², at least about 55 pmol/cm², at least about 60 pmol/cm², at least about 65 pmol/cm², at least about 70 pmol/cm², at least about 75 pmol/cm², at least about 80 pmol/cm², at least about 85 pmol/cm², at least about 90 pmol/cm², at least about 95 pmol/cm², at least about 100 pmol/cm², at least about 125 pmol/cm², at least about 150 pmol/cm², at least about 175 pmol/cm², at least about 200 pmol/cm², at least about 250 pmol/cm², at least about 300 pmol/cm², at least about 350 pmol/cm², at least about 400 pmol/cm², at least about 450 pmol/cm², at least about 500 pmol/cm², at least about 550 pmol/cm², at least about 600 pmol/cm², at least about 650 pmol/cm², at least about 700 pmol/cm², at least about 750 pmol/cm², at least about 800 pmol/cm², at least about 850 pmol/cm², at least about 900 pmol/cm², at least about 950 pmol/cm², at least about 1000 pmol/cm² or more.

Alternatively, the density of polynucleotide on the surface of the SNA is measured by the number of polynucleotides on the surface of a SNA. With respect to the surface density of polynucleotides on the surface of a SNA of the disclosure, it is contemplated that a SNA as described herein comprises from about 1 to about 100 oligonucleotides on its surface. In various embodiments, a SNA comprises from about 10 to about 100, or from 10 to about 90, or from about 10 to about 80, or from about 10 to about 70, or from about 10 to about 60, or from about 10 to about 50, or from about 10 to about 40, or from about 10 to about 30, or from about 10 to about 20 oligonucleotides on its surface. In further embodiments, a SNA comprises at least about 5, 10, 20, 30, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 polynucleotides on its surface.

Uses of SNAs in Gene Regulation/Therapy

In addition to serving a role in providing an oligonucleotide (e.g., an immunostimulatory oligonucleotide) and a tumor-associated antigen to a cell, it is also contemplated that in some embodiments, a SNA of the disclosure possesses the ability to regulate gene expression. In other words, in some aspects the disclosure provides a SNA comprising an oligonucleotide (e.g., an immunostimulatory oligonucleotide), a tumor-associated antigen, and an additional oligonucleotide designed to effect inhibition of target gene expression or perform some other regulatory function (e.g., target cell recognition). Accordingly, in some embodiments the disclosure provides methods for inhibiting gene product expression, and such methods include those wherein expression of a target gene product is inhibited by about or at least about 5%, about or at least about 10%, about or at least about 15%, about or at least about 20%, about or at least about 25%, about or at least about 30%, about or at least about 35%, about or at least about 40%, about or at least about 45%, about or at least about 50%, about or at least about 55%, about or at least about 60%, about or at least about 65%, about or at least about 70%, about or at least about 75%, about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 96%, about or at least about 97%, about or at least about 98%, about or at least about 99%, or 100% compared to gene product expression in the absence of a SNA. In other words, methods provided embrace those which results in essentially any degree of inhibition of expression of a target gene product.

The degree of inhibition is determined in vivo from a body fluid sample or from a biopsy sample or by imaging techniques well known in the art. Alternatively, the degree of inhibition is determined in a cell culture assay, generally as a predictable measure of a degree of inhibition that can be expected in vivo resulting from use of a specific type of SNA and a specific oligonucleotide.

In various aspects, the methods include use of an oligonucleotide which is 100% complementary to the target polynucleotide, i.e., a perfect match, while in other aspects, the oligonucleotide is at least (meaning greater than or equal to) about 95% complementary to the polynucleotide over the length of the oligonucleotide, about or at least about 90%, about or at least about 85%, about or at least about 80%, about or at least about 75%, about or at least about 70%, about or at least about 65%, about or at least about 60%, about or at least about 55%, about or at least about 50%, about or at least about 45%, about or at least about 40%, about or at least about 35%, about or at least about 30%, about or at least about 25%, about or at least about 20% complementary to the polynucleotide over the length of the oligonucleotide to the extent that the oligonucleotide is able to achieve the desired degree of inhibition of a target gene product. Moreover, an oligonucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure). The percent complementarity is determined over the length of the oligonucleotide. For example, given an inhibitory oligonucleotide in which 18 of 20 nucleotides of the inhibitory oligonucleotide are complementary to a 20 nucleotide region in a target polynucleotide of 100 nucleotides total length, the oligonucleotide would be 90 percent complementary. In this example, the remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleotides. Percent complementarity of an inhibitory oligonucleotide with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

Accordingly, methods of utilizing a SNA of the disclosure in gene regulation therapy are provided. This method comprises the step of hybridizing a polynucleotide encoding the gene with one or more oligonucleotides complementary to all or a portion of the polynucleotide, the oligonucleotide being the additional oligonucleotide of a composition as described herein, wherein hybridizing between the polynucleotide and the oligonucleotide occurs over a length of the polynucleotide with a degree of complementarity sufficient to inhibit expression of the gene product. The inhibition of gene expression may occur in vivo or in vitro.

The oligonucleotide utilized in the methods of the disclosure is either RNA or DNA. The RNA can be an inhibitory RNA (RNAi) that performs a regulatory function, and in various embodiments is selected from the group consisting of a small inhibitory RNA (siRNA), an RNA that forms a triplex with double stranded DNA, and a ribozyme. Alternatively, the RNA is microRNA that performs a regulatory function. The DNA is, in some embodiments, an antisense-DNA.

In various embodiments, the target polynucleotide encodes programmed death 1 (PD-1) or programmed death-ligand 1 (PD-L1).

Use of SNAs in Immune Regulation

Toll-like receptors (TLRs) are a class of proteins, expressed in sentinel cells, that plays a key role in regulation of innate immune system. The mammalian immune system uses two general strategies to combat infectious diseases. Pathogen exposure rapidly triggers an innate immune response that is characterized by the production of immunostimulatory cytokines, chemokines and polyreactive IgM antibodies. The innate immune system is activated by exposure to Pathogen Associated Molecular Patterns (PAMPs) that are expressed by a diverse group of infectious microorganisms. The recognition of PAMPs is mediated by members of the Toll-like family of receptors. TLR receptors, such as TLR 4, TLR 8 and TLR 9 that respond to specific oligonucleotide are located inside special intracellular compartments, called endosomes. The mechanism of modulation of TLR 4, TLR 8 and TLR9 receptors is based on DNA-protein interactions.

Synthetic immunostimulatory oligonucleotides that contain CpG motifs that are similar to those found in bacterial DNA stimulate a similar response of the TLR receptors. Therefore immunomodulatory oligonucleotides have various potential therapeutic uses, including treatment of immune deficiency and cancer.

Down regulation of the immune system would involve knocking down the gene responsible for the expression of the Toll-like receptor. This antisense approach involves use of SNAs conjugated to specific antisense oligonucleotide sequences to knock down the expression of any toll-like protein.

Accordingly, methods of utilizing SNAs for modulating toll-like receptors are disclosed. The method either up-regulates or down-regulates the Toll-like-receptor through the use of a TLR agonist or a TLR antagonist, respectively. The method comprises contacting a cell having a toll-like receptor with a SNA of the disclosure. The toll-like receptors modulated include toll-like receptor 1, toll-like receptor 2, toll-like receptor 3, toll-like receptor 4, toll-like receptor 5, toll-like receptor 6, toll-like receptor 7, toll-like receptor 8, toll-like receptor 9, toll-like receptor 10, toll-like receptor 11, toll-like receptor 12, and toll-like receptor 13.

Compositions. The disclosure includes compositions that comprise a pharmaceutically acceptable carrier and a cell having a spherical nucleic acid (SNA) contained therein, wherein the cell is obtained from an individual and the SNA comprises a nanoparticle, an oligonucleotide on the surface of the nanoparticle, and an antigen. In some embodiments, the composition is an antigenic composition. The term “carrier” refers to a vehicle within which the SNA is administered to a mammalian subject. The term carrier encompasses diluents, excipients, adjuvants and combinations thereof. Pharmaceutically acceptable carriers are well known in the art (see, e.g., Remington's Pharmaceutical Sciences by Martin, 1975).

Exemplary “diluents” include sterile liquids such as sterile water, saline solutions, and buffers (e.g., phosphate, tris, borate, succinate, or histidine). Exemplary “excipients” are inert substances include but are not limited to polymers (e.g., polyethylene glycol), carbohydrates (e.g., starch, glucose, lactose, sucrose, or cellulose), and alcohols (e.g., glycerol, sorbitol, or xylitol).

Adjuvants are include but are not limited to emulsions, microparticles, immune stimulating complexes (iscoms), LPS, CpG, or MPL.

Adoptive Cell Therapy. The disclosure includes methods of treating an individual in need of adoptive cell therapy, comprising administering to the individual an effective amount of a composition of the disclosure. Adoptive cell therapy involves isolating cells from an individual, expanding the cells ex vivo, and infusing the cells back to the patient.

For adoptive cell therapy using antigen-specific cells, cell doses in the range of about 10⁶ to about 10¹⁰ (e.g., about 10⁹) are typically infused. Upon administration of the cells into the subject and subsequent differentiation, the cells are induced that are specifically directed against one specific antigen (e.g., a tumor-associated antigen). The cells of the presently disclosed subject matter can be administered by any methods known in the art, including, but not limited to, pleural administration, intravenous administration, subcutaneous administration, intranodal administration, intratumoral administration, intrathecal administration, intrapleural administration, intraperitoneal administration, and direct administration to the thymus. In certain embodiments, the cells are intravenously administered to the subject in need.

The presently disclosed subject matter provides various methods of using the cells (e.g., T cells) expressing a tumor-associate antigen. For example, the presently disclosed subject matter provides methods of reducing tumor burden in a subject. In one non-limiting example, the method of reducing tumor burden comprises administering an effective amount of the presently disclosed composition to the subject, thereby inducing tumor cell death in the subject. The presently disclosed cell can reduce the number of tumor cells, reduce tumor size, and/or eradicate the tumor in the subject. Non-limiting examples of an individual in need of a composition of the disclosure include those individuals suffering from a cancer selected from the group consisting of prostate, breast, melanoma, and lung cancer.

The presently disclosed subject matter also provides methods of increasing or lengthening survival of a subject having cancer. In some embodiments, the method of increasing or lengthening survival of a subject having cancer comprises administering an effective amount of a composition of the disclosure to the subject, thereby increasing or lengthening survival of the subject. The method can reduce or eradicate tumor burden in the subject.

Methods of inducing an immune response. The disclosure includes methods for eliciting an immune response in a subject in need thereof, comprising administering to the subject an effective amount of a composition or vaccine of the disclosure.

The immune response raised by the methods of the present disclosure generally includes an antibody response, preferably a neutralizing antibody response, preferably a protective antibody response. The immune response generated by a composition as disclosed herein is directed against, and preferably ameliorates and/or neutralizes and/or reduces the tumor burden of cancer. Methods for assessing antibody responses after administration of a composition of the disclosure (immunization or vaccination) are known in the art and/or described herein. In some embodiments, the immune response comprises a T cell-mediated response (e.g., peptide-specific response such as a proliferative response or a cytokine response). In preferred embodiments, the immune response comprises both a B cell and a T cell response. Antigenic compositions can be administered in a number of suitable ways, such as intramuscular injection, subcutaneous injection, intradermal administration and mucosal administration such as oral or intranasal. Additional modes of administration include but are not limited to intranasal administration, and oral administration.

Antigenic compositions may be used to treat both children and adults. Thus a subject may be less than 1 year old, 1-5 years old, 5-15 years old, 15-55 years old, or at least 55 years old. Preferred subjects for receiving the vaccines are the elderly (e.g., >55 years old, >60 years old, preferably >65 years old), and the young (e.g., <6 years old, 1-5 years old, preferably less than 1 year old).

Administration can involve a single dose or a multiple dose schedule. Multiple doses may be used in a primary immunization schedule and/or in a booster immunization schedule. In a multiple dose schedule the various doses may be given by the same or different routes, e.g., a parenteral prime and mucosal boost, or a mucosal prime and parenteral boost. Administration of more than one dose (typically two doses) is particularly useful in immunologically naïve subjects or subjects of a hyporesponsive population (e.g., diabetics, or subjects with chronic kidney disease). Multiple doses will typically be administered at least 1 week apart (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, or about 16 weeks). Preferably multiple doses are administered from one, two, three, four or five months apart. Antigenic compositions of the present disclosure may be administered to patients at substantially the same time as (e.g., during the same medical consultation or visit to a healthcare professional) other vaccines.

In general, the number of cells (comprising SNAs as disclosed herein) in each dose of the antigenic composition is selected as an amount effective to induce an immune response in the subject, without causing significant, adverse side effects in the subject. Preferably the immune response elicited is a neutralizing antibody, preferably a protective antibody response. The number of cells to be administered is, in various embodiments, about 100,000 to 1×10⁶, or about 500,000 to about 1×10⁶, or about 1×10⁶ to about 5×10⁶, or about 1×10⁶ to about 1×10¹⁰.

Articles of Manufacture and Kits. The disclosure additionally includes articles of manufacture and kits comprising a composition described herein. In some embodiments, the kits further comprise instructions for measuring antigen-specific antibodies. In some embodiments, the antibodies are present in serum from a blood sample of a subject immunized with a composition comprising an SNA of the disclosure.

As used herein, the term “instructions” refers to directions for using reagents contained in the kit for measuring antibody titer. In some embodiments, the instructions further comprise the statement of intended use required by the U.S. Food and Drug Administration (FDA) in labeling in vitro diagnostic products.

The following examples illustrate various embodiments contemplated by the present disclosure. The figures provided herein are exemplary in nature and are in no way intended to be limiting.

EXAMPLES Example 1 Materials and Methods

Mice and reagents. C57BL/6 WT, and Pmel-1 mice were purchased from Jackson Laboratory. OT-1 Rag1−/− mice were purchased from Taconic. Dr. Hans Schreiber (University of Chicago) provided B16F10 cell line. All the cell lines were routinely tested for mycoplasma infections by culture and DNA stain, and maintained in complete medium composed of RPMI 1640 with 10% FBS. All animal experiments were approved by institutional animal use committees of Northwestern University. All mAbs were obtained from eBiosciences and BioLegend. Proliferation dye eFluor 450 was from eBiosciences.

SNAs, oligonucleotides, and antigens. For SNA and admix controls the peptides hgp10025-33 and OVA257-264 were purchased from Anaspec. Oligonucleotides were synthesized using automated solid support phosphoramidite synthesis. SNAs were synthesized as described [Banga et al., Journal of the American Chemical Society. 2014; 136(28):9866-9]. SNAs consisted of cholesterol-terminated CpG oligonucleotides (3′-TCCATGACGTTCCTGACGTT-5′ (SEQ ID NO: 1)) with phosphorthioate internucleotide linkages, adsorbed onto 50-nm diameter DOPC (di-oleoylphosphocholine) liposomes prepared by membrane extrusion. Peptide antigens were incorporated into SNA structures by encapsulation within liposomes. SNAs were purified from unadsorbed oligonucleotide and from unincorporated antigen by tangential flow filtration, or filtration through polycarbonate filters and resuspension in PBS.

Analysis of cells by flow cytometry. All samples were initially incubated with 2.4G2 to block antibody binding to Fc receptors. Single-cell suspensions were stained with 1 μg of relevant mAbs and then washed twice with cold PBS. Cytokine detection was performed by restimulating 1-3×10⁶ cells in single cell suspensions in a cocktail of 17 μg/mL of Brefeldin A, 50 ng/mL of PMA, and 1 μg/mL of ionomycin for 4 hours in RPMI 1640 supplemented with 10% FBS prior to following manufacturer protocol for intracellular cytokine staining (BD Biosciences).

In vivo tumor challenges and T chaperone therapy. B16F10 or LLC1-OVA (1×10⁶) in suspension were injected s.c. into the rear right flank of mice. On day 8 or when tumors reached approximately 150 mm³, T chaperones were transferred iv. in a volume of 200 μl of PBS. Prior to infusion, T chaps were generated from naïve T-cells by incubated with controls (mixtures of antigen and oligonucleotide) or SNAs overnight, washed three times and immediately transferred. In all experiments the size of tumor was determined at 2-3 day intervals. Tumor volumes were measured along orthogonal axes (a, b, and c) and calculated as abc/2.

In vitro activation of T chaperones. In all experiments CD8⁺ PMEL T cells were selected using EasySep™ CD8⁺ T cell positive selection kit II from STEMCELL technologies. For overnight activation, 0.5×10⁶ T cells were plated in 200 μl of RPMI 1640 supplemented with 10% FBS. gp100 peptide was added at a final concentration of 4 μg/ml and ova-I at 0.06 μg/ml as either free peptide or encapsulated in the core of SNAs. CpG stimulating oligonucleotide or GpC control oligonucleotide was given at 1 μM final concentration as either free linear form or in SNA form. In all experiments cells were washed with PBS three times prior to re-plating or transfer into animal.

Statistical analysis. Mean values were compared using an unpaired Student's two-tailed t test. Probability values >0.05 were considered non-significant.

Results

T chaperones (T chaps) possess ability to induce T cell priming in vivo. One goal of immunotherapy is to activate endogenous antigen specific CD8⁺ T cells to kill cancer cells. The approach disclosed herein is the treatment of T-cells ex vivo with SNAs, to load T-cells with immunestimulatory oligonucleotide and tumor-associated antigen, and to use these cells, “T chaperones” (T chaps), to activate endogenous antigen specific CD8⁺ T cells. Previous data showed that T chaps can activate naïve CD8⁺ T cells in vitro, so experiments disclosed herein were designed to demonstrate these effects in vivo. Using an in vivo priming assay, it was found that both T cell chaperones generated by treating naïve T-cells with free CpG and free peptide admix (T chapcontrol) and those generated by treatment with IS-NAs (T chap_(ISNA)) were able to induce proliferation of naïve responder cells in both spleen and lymph nodes (FIG. 5 A,B). However, only T chap_(ISNA) were able to induce the anti-tumor molecule IFN-γ production from naïve PMEL responder T cells in the lymph nodes (FIG. 5A). This correlates well with anti-tumor data and increased IFN-γ production by tumor infiltrating T cells in T chap_(ISNA) treated mice.

T chap_(ISNA) retain long term killing functions. The long term effects of SNAs on T cell effector functions was then tested. A control group consisting of T chaps generated by an overnight stimulation by a variety of immunestimulatory treatments (shown in FIG. 6A was used for comparison with T chaps that were evaluated 4 days following their generation with the same immunestimulatory conditions. The four day experimental group was generated by incubating CD8⁺ PMEL T cells overnight, washing them the following day and then re-plating in fresh medium in the absence of additional SNAs or control materials for four days. The control group was prepared one day in advance of evaluation by plating with B16 target tumor cells. The results showed that T chap_(ISNA) have better killing function compared to all controls and also have a durable effector response. This suggests a long term programming for killing function initiated by SNAs (FIG. 6A).

These effects were also seen in in vivo tumor challenges, in the comparison of admix of CpG and antigen and IS-SNAs in the generation of T chaps for adoptive cellular therapy. Using two models, B16 melanoma and LLC1-OVA lung carcinoma (FIG. 6B, 7A) it was observed that T chap_(ISNA) are superior to conventional activation and to admix activation to control both models. In the tumor microenvironment it was found that T chap_(ISNA) treatment enhanced the accumulation of polyfunctional tumor infiltrating CD8⁺ T cells as indicated by comparing the number of IFN-γ⁺ TNF-α⁺ double positive CD8⁺ T cells.

SNA structure induces T chap exosomal antigen transfer for bystander priming. The goal of the experiments that led to the collection of data in FIG. 8 was to gain insight into the mechanism of T chap-induced priming. Whether T chaps were able to transfer antigen to dendritic cells through soluble factors was tested first. T chaps and two types of bystander recipient cells (T-cells and DCs) were separated using a transwell system where T chaps were placed in the top well and separated by a membrane with 0.4 μm pores. This experiment tested for the possibility that T cells are able to pass these soluble factors to other (naïve) T cells and endow them with the ability to become antigen presenting T cells (T-APC). After overnight transwell incubation the recipient cells, the recipient T-APC and DCs were able to prime CD45.1⁺ PMEL T (FIG. 8A), showing that exosomal transfer of antigen to both T cells and DCs is one way in which T chaps induce priming.

Whether exosomes were able to directly induce T cells priming was tested next. CD8⁺PMEL T cells were activated and exosomes were collected after four days of culture. The exosomes were then directed plated with naïve e450 stained PMEL responder cells, and activation and proliferation were studied after four days. It was found that only exosomes derived from T chap_(ISNA) induced CD69 expression and proliferation (FIG. 8B). Whether T chap derived exosomes are capable of initiating T cell priming in vivo was then tested. After transfer of naïve e450 labeled CD8⁺ PMEL T cells, an equal amount of exosomes isolated from T chaps were also transferred iv. After five days, only the exosomes from T chap_(ISNA) induced proliferation of the naïve CD8⁺ PMEL T cells in inguinal lymph nodes (FIG. 8C).

CONCLUSIONS

Currently, adoptive T cell therapy in clinical settings involves lengthy, expensive and potentially dangerous use of transfection reagents to modify T cells. The three dimensional structure of SNAs allows for rapid cellular entry through receptor mediated endocytosis, bypassing the need for transfection reagents [Cutler et. al., Journal of the American Chemical Society. 2012; 134(3):1376-91]. Recently, it was discovered that non-phagocytic T cells can rapidly and efficiently uptake spherical nucleic acids (SNAs) into the cytoplasm. The present disclosure used SNAs as a programming platform for the creation of cellular T cell chaperones, which possess unique anti-tumor capabilities. SNAs, as a cellular programming platform, have allowed for the successful creation of a multi-functional T cell chaperone that is capable of directly killing tumor cells, directly priming tumor antigen specific T cells, and transferring SNA materials (e.g., antigen and CpG oligonucleotide) to other immune cells via exosomes in vivo.

T chap_(ISNA) showed superior performance in these activities over T chaps prepared from linear CpG/free peptide admix. In all side by side experiments, T chap_(ISNA) dramatically outperformed their counter parts. In many cases, only T chap_(ISNA) groups exhibited any phenotype, and were the only T chap to retain durable effector function in vitro and in vivo. These data indicated that SNAs are a formulation of immunestimulatory oligonucleotide and antigen that are particularly capable of inducing a long term and stable programming of T chaps, allowing them to resist the suppressive mechanisms in the tumor microenvironment which induces exhaustion and anergy. This is supported by the accumulation of poly-functional T cells in tumor tissue capable of producing IFN-γ and TNF-α in T chap_(ISNA) tumor bearing animals. Additionally, T chap_(ISNA) were capable of initiating an anti-tumor cascade through exosomal antigen sharing. This feature allowed for the dissemination of SNA material in tumor draining lymph nodes and in the tumor microenvironment. It is important that dendritic cells (DCs), professional antigen presenting cells, were able to act as recipient bystander cells and gain priming function. This served as a way to amplify T chap_(ISNA) induced anti-tumor immunity. A final feature demonstrated here was the ability of T chap_(ISNA) to act as T-APCs by priming naïve antigen specific T cells. Antigen presentation by T cells is poorly studied, but according to the data provided herein, may play a previously underappreciated role in expanding antigen specific T cells.

By using two different tumor models with different model antigens (B16/gp100 and LLC1OVA/OVA-1) it was demonstrated herein that the effect of T chap_(ISNA) is not limited by a particular antigen. Due to the structure of SNAs, any antigen that is desired can be incorporated into the hollow core of SNAs for uptake by T cells and other immune cells. This means that this technology can be used against all forms of solid and liquid tumors. Furthermore, the effects disclosed herein were only tested using CpG oligonucleotides. As with antigen, SNAs can be customized to program cellular recipients by modification of hybridization style, length, and sequences. These include use of both DNA and RNA oligonucleotide sequences to target various toll-like receptors. 

1. A composition comprising a pharmaceutically acceptable carrier and a cell having a spherical nucleic acid (SNA) contained therein, wherein the cell is obtained from an individual and the SNA comprises a nanoparticle, an oligonucleotide on the surface of the nanoparticle, and an antigen.
 2. The composition of claim 1, wherein the antigen is a prostate-specific antigen (PSA) peptide, mesothelin, glycoprotein 100 (gp100), prostate specific membrane antigen (PSMA), or prostatic acid phosphatase (PAP).
 3. The composition of claim 1, wherein the nanoparticle is a liposome.
 4. The composition of claim 3, wherein the liposome comprises a lipid selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC), 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DSPG), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE), cardiolipin, and lipid A.
 5. The composition of claim 3, wherein the oligonucleotide comprises a tocopherol, a cholesterol moiety, DOPE-butamide-phenylmaleimido, or lyso-phosphoethanolamine-butamide-pneylmaleimido.
 6. The composition of claim 1, wherein the oligonucleotide comprises RNA or DNA.
 7. The composition of claim 1, wherein the oligonucleotide comprises a sequence that is a toll-like receptor (TLR) agonist.
 8. The composition of claim 7, wherein the TLR is chosen from the group consisting of toll-like receptor 1 (TLR1), toll-like receptor 2 (TLR2), toll-like receptor 3 (TLR3), toll-like receptor 4 (TLR4), toll-like receptor 5 (TLR5), toll-like receptor 6 (TLR6), toll-like receptor 7 (TLR7), toll-like receptor 8 (TLR8), toll-like receptor 9 (TLR9), toll-like receptor 10 (TLR10), toll-like receptor 11 (TLR11), toll-like receptor 12 (TLR12), and toll-like receptor 13 (TLR13).
 9. The composition of claim 1, wherein the oligonucleotide comprises a CpG nucleotide sequence.
 10. The composition of claim 1, further comprising an additional oligonucleotide.
 11. The composition of claim 10, wherein the additional oligonucleotide comprises RNA or DNA.
 12. The composition of claim 11, wherein the RNA is a non-coding RNA.
 13. The composition of claim 12, wherein the non-coding RNA is an inhibitory RNA (RNAi).
 14. The composition of claim 13, wherein the RNAi is selected from the group consisting of a small inhibitory RNA (siRNA), a single-stranded RNA (ssRNA) that forms a triplex with double stranded DNA, and a ribozyme.
 15. The composition of claim 12, wherein the RNA is a microRNA.
 16. The composition of claim 11, wherein the DNA is antisense-DNA.
 17. The composition of claim 1, wherein the nanoparticle has a diameter of 50 nanometers or less.
 18. The composition of claim 1 comprising about 10 to about 80 double stranded oligonucleotides.
 19. The composition of claim 18 comprising 70 double stranded oligonucleotides.
 20. The composition of claim 1, wherein the cell is a T-cell, a natural killer (NK) cell, a B-cell, a macrophage, a dendritic cell, or a combination thereof.
 21. The composition of claim 1, wherein the antigen is encapsulated in the nanoparticle.
 22. The composition of claim 1, wherein the antigen is on the surface of the nanoparticle.
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 25. A vaccine comprising the composition of claim 1, and an adjuvant.
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